Small chemical moieties can and often do affect the environment and biological systems. These effects become astounding when it is realized that minute quantities of small moieties are involved. Moreover, the presence or absence of low concentrations of small moieties in the environment can have long term consequences. Lead in water and gasoline bear witness. Minute quantities of metallic cations and small organic molecules can regulate, influence, change or toxify the environment or biological systems.
The detection, removal, addition or neutralization of such minute quantities constitutes a focal point for continued research in many fields. For example, many efforts have been made to detect and remove minute, toxic amounts of heavy metal ions such as mercury from the environment. The efforts often have not been successful or economical for widespread application. On the other hand, minute concentrations of other heavy metals are important for the proper function of biological organisms. Zinc, for example, plays a major role in wound healing. The function of magnesium in plant photosynthesis is another.
Small moieties also exhibit dual roles. Mercury is used in diuretics, topical anti-bacterial agents, skin antiseptics, ointments, and in chemical manufacturing operations. Yet when ingested by mammals, such as from drinking water, it is highly toxic in very small amounts. Hence, detection and quantification of minute concentrations of heavy metals in drinking water and other media would serve exploratory, safety and regulatory goals.
Small organic molecules such as cleaning fluids (e.g. trichloroethylene), as well as many pesticides and herbicides have small business, agricultural and industrial applications. The former are used in processing, formulating, cleaning and purifying while the latter retard infestation by vermin, insects and undesired plants. However, these molecules also find their way into ground water and subsequently contaminate water used for consumption, agricultural and industrial purposes. Hence, efficient and accurate identification of minute concentrations of small organic molecules in drinking water or other media would be an important step toward their control.
Cosmetic formulations, perfumes, and other proprietary products often contain minute levels of certain small organic compounds. The appropriate selection and mixture of these compounds is the secret of the perfumer's art. Hence, determination of the concentrations and identities of these compounds could serve as a means for cosmetic control or for cosmetic design.
Many foods contain minute quantities of small organic compounds. These compounds contribute to the flavor notes and odor of the foods. For example, ethyl butyrate and limonene contribute to the fresh flavor notes so characteristic of freshly squeezed orange juice. Hence, determination of the concentrations and identities of such compounds within foods and the isolation and purification of the same would help advance food design and screening.
Removal of minute quantities of small moieties from biological or inanimate systems carries many implications. Sea water contains minute concentrations of gold and platinum. Economic removal and refining of these noble metals from sea water could be rewarding. Metal, in particular, lead poisoning has long had serious life-threatening and debilitating consequences. The ability to easily detect traces of toxic metals on site and in the field and to selectively remove toxic metals from fluids and tissues of children and other people, contaminated with trace amounts of such metals, should provide major steps in controlling and eliminating these problems. Finally, nuclear contaminants such as radioactive strontium, cobalt and others can endanger the population. Selective removal of these radioactive isotopes from the fluids and tissues of people so contaminated could avoid radiation sickness.
It would, therefore, be highly desirable to identify and control minute quantities of helpful or harmful small moieties in aqueous biological or inanimate systems. In most contexts, however, the detection, removal, addition or neutralization of small moieties is a difficult, time consuming, expensive and often unfeasible if not impossible task. Other compounds often mimic the small moieties and interference with measurements will result. Moreover, the detection methods employed today are usually not sufficiently sensitive at the minute quantities under consideration here. Consequently, it is desirable to develop reliable, quick and economic methods which can be used in the lab or the field for accurately identifying and controlling minute quantities of small moieties in aqueous systems.
Antibodies would seem to be uniquely suited for this task. Their high degree of specificity for a known antigen would avoid the interference caused by contaminants. Their sensitivity in the picomolar or lower range would accurately and efficiently target and detect the minute levels.
Monoclonal antibodies, of course, come to mind as especially suited agents for practice of this technique. Since Kohler and Milstein published their article on the use of somatic cell hybridization to produce monoclonal antibodies (Nature 256:495 (1974)), immunologists have developed many monoclonal antibodies which strongly and specifically immunoreact with antigens.
Notwithstanding this suggestion, the conventional understanding about immunology teaches that antibodies against small moieties cannot be developed. The mammal immunization step, which is key for the production of monoclonal antibodies, requires a molecule that is large enough to cause antigenic reaction. Medium sized molecules (haptens), which are not of themselves immunogenic, can induce immune reaction by binding to an immunogenic carrier. Nevertheless, immunologists view small molecules such as metallic cations and small organic molecules as not large or structurally complex enough to elicit an antibody response. One theory appears to hold that electron rich rings such as those associated with benzene and carbohydrates are needed at a minimum to cause immunogenicity. V. Butler, S. Beiser, Adv. Immunol., 17, 255 (1973). The molecular size and complexity of an inorganic or organic small moiety is thought to be insufficient for eliciting an antibody response. To date, therefore, no monoclonal antibodies which immunoreact with small moieties per se have been reported in the literature.
Several immunologists have reported production of monoclonal antibodies to metallic ion chelates. For example, in U.S. Pat. No. 4,722,892, monoclonal antibodies are disclosed which immunoreact with a complex of a chelating agent, such as ethylene diamine tetracetate (EDTA), and a heavy metal such as indium. In EPO Patent Application 0235457, monoclonal antibodies that immunoreact with a chelate of gold cyanate and carbonate coating are disclosed. In these instances, however, the monoclonal antibodies bind with the metal chelate complex rather than the bare metallic ion itself. Disadvantages of these methods include: the complicated reagents involved in detection, lack of simple tests that discriminate among antigens, cross-reactivity with chelates of other antigens and cross-reactivity with the chelate itself.
Other instances of monoclonal antibody combinations with metals involve metal tags. The metal chelates are bound to the antibody at a site remote from the antigen binding site or sites. The metal or metal chelate is not the antigen. Instead, it is a tag to indicate the presence of the monoclonal antibody when it reacts with its specific antigen. See for example, V. P. Torchilian et al., Hybridoma, 6, 229 (1987); and C. F. Meares, Nuclear Medical Biology, 13, 311-318 (1986).
It is therefore, an object of the invention to develop monoclonal antibodies and fragments thereof that immunoreact with small moieties per se. It is another object of the invention to develop methods for detecting or neutralizing small moieties within, adding small moieties to, or removing small moieties from biological or inanimate systems through the use of the monoclonal antibodies and fragments thereof. Further objects include development of hybridomas which produce the monoclonal antibodies, development of immunogen compounds for generation of antibody reactivity to the small moieties and development of methods for production of antibody fragments. Yet another object is the development of monoclonal antibodies and fragments thereof that are capable of discriminating very similar small moieties.